Myxoma virus mutants for cancer treatment

ABSTRACT

Myxoma viruses that are deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M 136, M-T4 and M-T7 are useful for treating cancer.

BACKGROUND OF THE INVENTION

The use of certain genetically modified myxoma viruses for treatingcancer is disclosed in WO 04/078206 (Robarts Research Institute).

SUMMARY OF THE INVENTION

This invention relates to Myxoma viruses (MV) that are deficient in theactivity of a Myxoma virus protein selected from the group consisting ofM11L, M063, M136, M-T4 and M-T7. Such viruses are used in a method forand in the manufacture of a medicament for, inhibiting a cancer cell,which method comprises administering to the cell an effective amount ofthe Myxoma virus. They are also used in a method for and in themanufacture of a medicament for, treating a human subject having cancer,comprising administering to the patient an effective amount of theMyxoma virus. This invention also provides a pharmaceutical compositioncomprising such Myxoma viruses and a pharmaceutically acceptablecarrier, as well as a kit comprising such Myxoma viruses andinstructions for treating a cancer patient.

DESCRIPTION OF THE FIGURES

FIG. 1. Endogenous activated Akt levels in human glioma cells

FIG. 2. Viral replication efficiency of the various vMyx-hrKOs andcontrols in human glioma cell lines.

FIG. 3. Secreted early and late viral gene expression indicates thatsome of the vMyx-hrKO are unable to transit from early to late geneexpression.

FIG. 4. Selected single step growth curves.

FIG. 5. Cell-based cytotoxicity assay

DETAILED DESCRIPTION OF THE INVENTION

WO 04/078206 (Robarts Research Institute) discloses the use of certaingenetically modified myxoma viruses for treating cancer. This inventionrepresents an advance by providing more specific modified myxoma virusesfor such uses. The techniques disclosed therein are applicable generallyto the myxoma viruses of this invention and the contents of WO 04/078206are incorporated herein by reference.

As used herein “deficient in the activity of” a given Myxoma virusprotein means that the virus has less of the activity in question thanwild-type Myxoma virus. “Substantially no activity” of a given viralprotein means that the virus has no detectable level of such activity.Examples of Myxoma viruses having substantially no activity of a givenviral protein include mutants in which the gene for such protein hasbeen deleted or otherwise knocked-out.

In accordance with this invention, any kind of cancer or cancer cell canbe inhibited or treated. In an embodiment of this invention, the cancercell is a mammalian cancer cell. In a more specific embodiment, thecancer cell is a human cancer cell. Examples of such human cancer cellsinclude gliomas.

It has been demonstrated that wild-type myxoma virus (vMyxgfp) canproduce a productive, long-lived infection, and destroy and clearimplanted tumor tissue when injected intratumorally into human gliomasimplanted in murine brains (Lun et al, 2005 Cancer Research65:9982-9990). As well, a screen of the NCI-60 reference collectionindicated that MV productively infects the majority (15/21) of humantumor cells tested (Sypula et al. 2004 Gene Ther. Mol. Biol. 8:103-114).To expand understanding of MV tropism in cancer cells, a series of humanglioma cells (U87, U118, U251, U343, U73) that were previously testedfor wild-type MV permissiveness were screened. These findings have beenextended in the following Examples by testing the infection andreplication of several MV viruses in which specific host range genes,identified as having a role in defining MV tropism in rabbit cells, havebeen deleted. These viruses are collectively referred to as host rangeknockouts (vMyx-hrKO). Variation was observed in the ability of variousvMyx-hrKOs to replicate and spread in the human glioma cells.vMyxT2(U251), vMyxT4KO (U87, U118, U251 and U373) and vMyxT5KO (U251,U373) exhibited some restriction in specific human gliomas. In contrastvMyx63KO and vMyx135KO appeared to replicate and kill more effectivelyin several of the gliomas.

The invention will be better understood by reference to the followingexamples, which illustrate but do not limit the invention describedherein.

EXAMPLES Example 1

Fifty micrograms of whole cell lysates were probed with antibodiesagainst phosphorylated Akt at positions threonine 308 (P-Akt T308) andserine 473 (P-Akt 5473) or total Akt. Based on the levels of activatedAkt U87 and U343 would be expected to be infectable and U118, U251 andU373 to be more resistant to MV infection. (FIG. 1).

Example 2

Virus stocks were titrated on the various glioma cells and control BGMKor RK13 cells. Virus titres, derived from the gliomas, were compared tothe control levels and a value of viral replication efficiency wasestimated. Based on these results none of the gliomas supported viralgrowth to the levels observed in the control lines. However some viruses(vMyx135KO, vMyx63KO and vMyx136KO) were more efficient than otherknockouts. As well, some glioma lines supported more replication (U87,U343 and U373). (FIG. 2)

Example 3

Various human gliomas were infected with a range of vMyx-hrKOs. Twentyhpi the infected supernatants were collected and concentrated 10×.Fifteen microlitres of concentrated sups were separated on a 12%SDS-PAGE gel and probed with anti-Serp1 (mAB; late MV gene product). Theblots were stripped and probed for early gene expression with anti-M-T7(pAB; early MV gene product). The results suggest that severalvMyx-hrKOs are restricted in their transit from early to late geneexpression including vMyxT2KO, vMyxT4KO and vMyxT5KO. And in two gliomalines (U87 and U37), vMyxT4KO does not even undergo early geneexpression. (FIG. 3).

Example 4

Cells were seeded in 48 well dishes and infected cells were collected atthe times indicated. Virus was released from the collected cell pelletsand titrated back onto BGMK cells. Although there was replication of thetested viruses, the best amplification appeared to occur in the U87 andU343 cells. (FIG. 4).

Example 5

The ability of the various vMyx-hrKOs and control viruses to have akilling effect in the panel of human gliomas was tested by acytotoxicity assay. The appropriate cells were seeded in 96 well dishesand 24 h later were infected with the viruses at various MOIs.Seventy-two hours post infection the infected cells were treated withthe WTS reagent (Roche) to measure cell viability. Colour changes weremeasured at 450 nm every 60 minutes for 4 hours. Uninfected controlwells were used to determine normal proliferation and a blank wellserved as a background control. (FIG. 5).

1-13. (canceled)
 14. A method for inhibiting a cancer cell comprisingadministering to the cell an effective amount of a Myxoma virus in whichthe gene for Myxoma virus protein M11L has been deleted or otherwiseknocked-out.
 15. The method of claim 4, wherein the cell is a mammaliancancer cell.
 16. The method of claim 4, wherein the cell is a humancancer cell.
 17. The method of claim 4, wherein the cell is a gliomacell.
 18. A method for treating a human subject having cancer,comprising administering to the subject an effective amount of a Myxomavirus in which the gene for Myxoma virus protein M11L has been deletedor otherwise knocked-out.
 19. The method of claim 18, wherein the canceris glioma.